From a blood safety perspective, transfusion-transmitted infections involving Babesia spp. have become increasingly problematic world-wide, with increased numbers of cases causing morbidity and mortality reported each year. Therefore the study of this blood-borne parasite should be considered as a priority for transfusion medicine. Our purpose is to identify key erythrocyte proteins and their parasite binding partners that mediate invasion of B. divergens into the human red blood cell (RBC), with the ultimate aim of exploiting parasite molecules found to play key roles in invasion as diagnostic and blood screening tools. The major goal of this application is to take advantage of the progress that has been made in the in vitro production of cultured red blood cells (cRBCs) by expansion of hematopoietic CD34+ cells, coincident with the progress made in the genetic characterization of blood group proteins, to manipulate cRBCs by shRNA mediated knock-down of specific transcripts and to engineer iPSCs by CRISPR/Cas9 knock-out of specific genes to decipher the receptors involved in Babesia invasion. Invasion of the human RBC is the central pathogenic step in the life- cycle of Babesia. Interruption of this step in the cycle would halt infection. This proposal describes how we will (1) generate antigen deficient cRBCs using use shRNA-mediated knockdown of gene expression in CD34+ cells and CRISPR/Cas9-mediated knockout of gene expression in iPSCs. After inducing ex vivo erythropoiesis, B. divergens invasion assays will be carried out in the engineered antigen deficient reticulocytes to identify RBC determinants important for parasite invasion (2) identify cognate parasite ligands using pull down assays and validate the receptor-ligand interactions biochemically and functionally and characterize their role in invasion in the parasite (3) determine the immunogenicity of these ligands using human and animal sera to design suitable ELISA assays for potential use in blood centers. This proposal uniquely will advance basic science methods to enhance production of mature cRBCs in the laboratory, provide insight into developmental regulation of antigen expression and provide a biological target for assessment of host molecules needed for Babesia invasion.